Temperature and Solvent Effects on the Europium(III)-catalyzed Hydrolysis of Bis-(nitrophenyl) Phosphate

The hydrolysis of bis-(nitrophenyl) phosphate (BNPP) as model for secondary phosphate esters is analyzed at six different concentrations of Eu(III) ions and four temperatures between 303 and 343 K. Eyring plots yield activation enthalpy parameters which with, e.g. [Eu 3+ ] between 0.40 and 10.0 mM drop from 130 to 74 kJ mol m 1 , respectively, with a relatively small drop in the opposing entropy contributions. The observed saturation profiles allow for the first time to evaluate the influence of j H and j S separately on the Michaelis-Menten values K M and k cat , showing that the catalytic metal-ion effects are largely due to changes in k cat and dominated by favorable j H changes. Preliminary studies of the solvent influence show a surprising difference between water mixtures with either ethanol or DMSO. With, e.g. 40% ethanol, one observes a doubling of the rate constant, with 40% DMSO an almost 10-fold rate decrease. In both cases, a linear correlation with the solvent polarity parameter E T is found.     

Probing potential medium effects on phosphate ester bonds using 18O isotope shifts on 31P NMR

Dipolar aprotic cosolvents, such as DMSO and acetonitrile, accelerate the rates of hydrolysis of phosphate monoester dianions. It has been speculated that the rate acceleration arises from the disruption of hydrogen bonding to the phosphoryl group. An aqueous solvation shell can stabilize the dianionic phosphoryl group by forming hydrogen bonds to the phosphoryl oxygens, whereas solvents such as DMSO are incapable of forming such bonds. It has been proposed that the loss of stabilization could result in a weakened P-OR ester bond, contributing to the observed faster rate of hydrolysis. Computational results support this notion. We have used the 18O-induced perturbation to the 31P chemical shift to ascertain whether solvent changes result in alterations to the P-O(R) bond. We have studied 16O18O-labeled methyl, ethyl, phenyl, p-nitrophenyl, diethyl p-nitrophenyl, triphenyl, and di-tert-butyl ethyl phosphate in the solvents water, methanol, chloroform, acetonitrile, dioxane, and DMSO. The results suggest no significant solvent-induced weakening of the phosphate ester bonds in any of the solvents tested, and this is unlikely to be a significant source for the acceleration of hydrolysis in mixed solvents.     

Thermodynamic origin of the increased rate of hydrolysis of phosphate and phosphorothioate esters in DMSO/water mixtures

The hydrolysis rates of the dianions of phosphate and phosphorothioate esters are substantially accelerated by the addition of polar aprotic solvents such as DMSO and acetonitrile. The activation barrier DeltaG is smaller due to a lower enthalpy of activation. The enthalpy of transfer of p-nitrophenyl phosphate (pNPP) and p-nitrophenyl phosphorothioate (pNPPT), from water to 0.6 (mol) aq DMSO (60 mol % water in DMSO) were measured calorimetrically. The enthalpies of activation for the hydrolysis reactions in the two solvents permitted the calculation of the enthalpy of transfer of the transition states. This transfer is thermodynamically favorable for both the reactants and the transition states but is more favorable for the transition states. In the case of pNPP, the enthalpy of transfer of the reactant is -23.9 kcal/mol, compared to -28.3 for the transition state. The difference is greater for pNPPT, where the enthalpy of transfer of the reactant is -23.2 kcal/mol and that for the transition state is -35.3. The results show that the reduced enthalpies of activation in both hydrolysis reactions arise not from a destabilization of the reactants in the mixed solvent, but from the fact that the enthalpy of transfer of the transition states to the mixed solvent is significantly more negative than the enthalpy of transfer of the reactants.     

Comparisons of phosphorothioate with phosphate transfer reactions for a monoester,diester, and triester: isotope effect studies

Phosphorothioate esters are sometimes used as surrogates for phosphate ester substrates in studies of enzymatic phosphoryl transfer reactions. To gain better understanding of the comparative inherent chemistry of the two types of esters, we have measured equilibrium and kinetic isotope effects for several phosphorothioate esters of p-nitrophenol (pNPPT) and compared the results with data from phosphate esters. The primary (18)O isotope effect at the phenolic group ((18)k(bridge)), the secondary nitrogen-15 isotope effect ((15)k) in the nitro group, and (for the monoester and diester) the secondary oxygen-18 isotope effect ((18)k(nonbridge)) in the phosphoryl oxygens were measured. The equilibrium isotope effect (EIE) (18)k(nonbridge) for the deprotonation of the monoanion of pNPPT is 1.015 +/- 0.002, very similar to values previously reported for phosphate monoesters. The EIEs for complexation of Zn(2+) and Cd(2+) with the dianion pNPPT(2-) were both unity. The mechanism of the aqueous hydrolysis of the monoanion and dianion of pNPPT, the diester ethyl pNPPT, and the triester dimethyl pNPPT was probed using heavy atom kinetic isotope effects. The results were compared with the data reported for analogous phosphate monoester, diester, and triester reactions. The results suggest that leaving group bond fission in the transition state of reactions of the monoester pNPPT is more advanced than for its phosphate counterpart pNPP, while alkaline hydrolysis of the phosphorothioate diester and triester exhibits somewhat less advanced bond fission than that of their phosphate ester counterparts.     

有机磷化合物的研究XXXIX. 溶剂性质对烷基膦酸-O, O-1,3-丙二酯及-O,O-1,4-丁二酯的碱性水解动力学的影响

本文报道烷基膦酸-O, O-1,3-丙二酯及O, O-1,4-丁二酯于50%二甲亚砜水溶液中的水解动力学, 考察了取代基结构对膦酸酯水解速率常数的影响。通过它们在50%的二甲亚砜水溶液和50%的二氧六环水溶液中碱性水解速率常数的比较, 说明在这些混合溶液中, 烷基的空间结构对水解速率常数的影响是近似平行的。同时, 正丙基膦酸-O, O-1,3-丙二酯于不同混合溶剂中碱性水解时, 溶剂分子的给质子接受质子的能力对水解反应过程有重要影响。     

Transition state differences in hydrolysis reactions of alkyl versus aryl phosphate monoester monoanions

Although aryl phosphates have been the subject of numerous experimental studies, far less data bearing on the mechanism and transition states for alkyl phosphate reactions have been presented. Except for esters with very good leaving groups such as 2,4-dinitrophenol, the monoanion of phosphate esters is more reactive than the dianion. Several mechanisms have been proposed for the hydrolysis of the monoanion species. (18)O kinetic isotope effects in the nonbridging oxygen atoms and in the P-O(R) ester bond, and solvent deuterium isotope effects, have been measured for the hydrolysis of m-nitrobenzyl phosphate. The results rule out a proposed mechanism in which the phosphoryl group deprotonates water and then undergoes attack by hydroxide. The results are most consistent with a preequilibrium proton transfer from the phosphoryl group to the ester oxygen atom, followed by rate-limiting P-O bond fission, as originally proposed by Kirby and co-workers in 1967. The transition state for m-nitrobenzyl phosphate (leaving group pK(a) 14.9) exhibits much less P-O bond fission than the reaction of the more labile p-nitrophenyl phosphate (leaving group pK(a) = 7.14). This seemingly anti-Hammond behavior results from weakening of the P-O(R) ester bond resulting from protonation, an effect which calculations have shown is much more pronounced for aryl phosphates than for alkyl ones.     

分子内反应与分子内催化III. 溶剂效应对2-(2'-苯并咪唑基)乙 酸乙酯及3- (2'-苯并咪唑基)丙酸乙酯碱性水解的影响

测定了2-(2'-苯并咪唑基)乙酸乙酯及3-(2'-苯并咪唑基)丙酸乙酯在DMSO-水、1,4-二氧六环-水混合溶剂体系中的碱性水解动力学。随着DMSO-H~2O混合溶剂中DMSO含量增加,两种酯水解表观速率常数分别呈现出不规则的钟形变化。实验结果与我们所提出的酯水解历程中既包括分子间氢氧根离子特殊碱催化又包括分子内苯并咪唑基一般碱催化两种催化方式相符合。     

Activating water: important effects of non-leaving groups on the hydrolysis of phosphate triesters

The high rate of spontaneous hydrolysis of tris-2-pyridyl phosphate (TPP) is explained by the activating effects of the non-leaving ("spectator") groups on P-OAr cleavage, and not by intramolecular catalysis. Previous work on phosphate-transfer reactions has concentrated on the contributions to reactivity of the nucleophile and the leaving group, but our results make clear that the effects of the non-leaving groups on phosphorus can be equally significant. Rate measurements for three series of phosphate triesters showed that sensitivities to the non-leaving groups are substantial for spontaneous hydrolysis reactions, although significantly smaller for reactions with good nucleophiles. There are clear differences between triaryl and dialkyl aryl triesters in sensitivities to leaving and non-leaving groups with the more reactive triaryl systems showing lower values for both β(LG) and β(NLG). Intramolecular catalysis of the hydrolysis of TPP by the neighbouring pyridine nitrogens is insignificant, primarily because of their low basicity.     

Solvent effects and alkali metal ion catalysis in phosphodiester hydrolysis

The kinetics of the alkaline hydrolysis of bis(p-nitrophenyl) phosphate (BNPP) have been studied in aqueous DMSO, dioxane, and MeCN. In all solvent mixtures the reaction rate steadily decreases to half of its value in pure water in the range of 0-70 vol % of organic cosolvent and sharply increases in mixtures with lower water content. Correlations based on different scales of solvent empirical parameters failed to describe the solvent effect in this system, but it can be satisfactorily treated in terms of a simplified stepwise solvent-exchange model. Alkali metal ions catalyze the BNPP hydrolysis but do not affect the rate of hydrolysis of neutral phosphotriester p-nitrophenyl diphenyl phosphate in DMSO-rich mixtures. The catalytic activity decreases in the order Li+ > Na+ > K+ > Rb+ > Cs+. For all cations except Na+, the reaction rate is first-order in metal ion. With Na+, both first- and second-order kinetics in metal ions are observed. Binding constants of cations to the dianionic transition state of BNPP alkaline hydrolysis are of the same order of magnitude and show a similar trend as their binding constants to p-nitrophenyl phosphate dianion employed as a transition-state model. The appearance of alkali metal ion catalysis in a medium, which solvates metal ions stronger than water, is attributed to the increased affinity of cations to dianions, which undergo a strong destabilization in the presence of an aprotic dipolar cosolvent.     

Hydrolysis of cyclic phosphates by ribonuclease A: a computational study using a simplified ab initio quantum model

The second step in the enzyme-catalyzed hydrolysis of phosphate esters by ribonuclease A (RNase A) was studied using an ab initio quantum-based model of the active site including constrained parts of three critical residues, His-12, His-119, and Lys-41, and a small substrate. The competition between release of the cyclic phosphate intermediate and subsequent hydrolysis following transphosphorylation was explored to determine the electronic factors that contribute to preferential intermediate product release observed experimentally. The structural and energetic results obtained at both the RHF and MP2 levels reveal several contributing factors consistent with experimental observation. Although the intrinsic electronic effects tend to favor hydrolysis slightly with an overall activation free energy of approximately 70 kJ mol(-1), entropic and environmental effects favor release of the cyclic phosphate intermediate over hydrolysis. Exploration of the second, hydrolysis step also revealed interesting similarity with the transphosphorylation step, including the observation of autocatalysis by the substrate. Moreover, both steps of the overall RNase A reaction reveal multiple pathways involving proton transfers to sites of similar proton affinities. The anionic phosphate in both steps can act as a stable proton binding site as protons are moved around the active site throughout the progress of the reaction. These results suggest autocatalysis may be representative of more general behavior in enzymes containing highly charged substrates, especially phosphates.     

Structural, kinetic, and theoretical studies on models of the zinc-containing phosphodiesterase active center: medium-dependent reaction mechanisms

Dinuclear zinc(II) complexes [Zn(2)(bpmp)(mu-OH)](ClO(4))(2) (1) and [Zn(2)(bpmp)(H(2)O)(2)](ClO(4))(3) (2) (H-BPMP=2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol) have been synthesized, structurally characterized, and pH-driven changes in metal coordination observed. The transesterification reaction of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) in the presence of the two complexes was studied both in a water/DMSO (70:30) mixture and in DMSO. Complex 2 was not reactive whereas for 1 considerable rate enhancement of the spontaneous hydrolysis reaction was observed. A detailed mechanistic investigation by kinetic studies, spectroscopic measurements ((1)H, (31)P NMR spectroscopy), and ESI-MS analysis in conjunction with ab initio calculations was performed on 1. Based on these results, two medium-dependent mechanisms are presented and an unusual bridging phosphate intermediate is proposed for the process in DMSO.     

Solvolysis of Tris-p-nitrophenyl-phosphate in aqueous and reverse micelles

The widespread use of toxic phosphates and phosphonates as insecticides, and their use as chemical weapons, has led to investigation of fast detoxification and decontamination methods. Micelles, microemulsions, cyclodextrines and liposomes have been used to accelerate phosphate ester decomposition by nucleophiles. Here, hydrolysis, methanolysis and hexanolysis of Tris-p-nitrophenyl phosphate (TNPP), a model for reactive phosphate esters, were studied in homogeneous phase, aqueous and reverse micelles. Kinetic micellar effects were quantitatively analyzed using pseudo-phase models. TNPP hydrolysis was catalyzed by cetyltrimethylammonium chloride (CTAC), cetyltrimethylammonium bromide (CTAB), and hexadecylammonium propanesulfonate (HPS), micelles by factors of five, CTAC, and three, CTAB, HPS, respectively. The calculated rate constants for spontaneous and acetate-catalyzed hydrolysis in the micellar phase were significantly higher than those in the aqueous phase. While in water and in methanol the effect of the acetate cation was negligible, the catalytic efficiency of acetate for hexanolysis depended on the nature of the cation with the K⁺ salt being ca. 20 times more efficient than the tetraethylammonium salt in non-polar solvents. Sodium dodecylsulfate, SDS, micelles inhibited TNPP hydrolysis by a factor of eigth. Reverse micelles of CTAB in n-hexanol/isooctane (10:90, v/v) did not catalyze TNPP hydrolysis, but changed the bis-p-nitrophenyl phosphate/hexyl-bis-p-nitrophenylphosphate product ratio depending of CTAB concentration and water/detergent ratio.     

The thermodynamics of phosphate versus phosphorothioate ester hydrolysis

Phosphorothioate esters are phosphate esters in which one of the nonbridging oxygen atoms has been replaced by sulfur. In the comparative hydrolysis reactions of phosphorothioate and phosphate esters, the sulfur substitution accelerates the rates of the monoesters while slowing the rates of diesters and of triesters. Previously measured enthalpies and entropies of activation for the hydrolysis reactions of the monoesters, p-nitrophenyl phosphate and p-nitrophenyl phosphorothioate, were compared to the activation parameters measured herein for the diesters, ethyl p-nitrophenyl phosphate and ethyl p-nitrophenyl phosphorothioate, and the triesters, diethyl p-nitrophenyl phosphate and diethyl p-nitrophenyl phosphorothioate. A consistent trend of a greater DeltaH++ for the phosphorothioate analogue was found in all three classes of ester. In the monoester case, a more positive DeltaS++ arising from a mechanistic difference (D(N) + A(N) for the phosphorothioate versus A(N)D(N) for the phosphate) compensates, resulting in a lower DeltaG++ for the phosphorothioate monoester. Spectroscopic investigations indicate there is no significant difference in bond order to the leaving group in phosphates, as compared to their phosphorothioate analogues, ruling this out as a contribution to the consistently higher enthalpies of activation.     

Simplified speciation and improved phosphodiesterolytic activity of hydroxo complexes of trivalent lanthanides in aqueous DMSO

Potentiometric titrations of La(III), Nd(III), and Eu(III) perchlorates by Me 4N(OH) in 80% vol aq DMSO revealed formation of predominantly mononuclear complexes M(OH)n(3- n) (n = 1, 2, or 3) and a single binuclear complex M2(OH)(5+). Kinetics of the cleavage of two phosphate diesters, bis (4-nitrophenyl) phosphate (BNPP) and 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP), and a triester, 4-nitrophenyl diethyl phosphate (paraoxon), were studied as a function of metal and Me4N(OH) concentrations in the same medium. Rate of BNPP cleavage is second-order in metal and is proportional to the product of concentrations of M(OH)2(+) and M(OH)3 species. Rate of HPNPP cleavage is proportional to [M(OH)3](3) for La(III) and Nd(III) and to [M(OH)3](2) for Eu(III). Proposed mechanism for BNPP hydrolysis involves formation of M2(OH)5(diester) intermediate followed by intramolecular nucleophilic attack of hydroxide anion on the phosphoryl group of the substrate. Proposed mechanism for HPNPP cleavage involves formation of M3(OH)9(diester)(-) or M2(OH)6(diester)(-) intermediates followed by the general base-assisted intramolecular cyclization of HPNPP. The latter mechanism is supported by observation of the solvent kinetic isotope effect k H/kD = 2.9 for Eu(III) catalyzed HPNPP cleavage. The efficiency of catalysis in 80% DMSO is much higher than in water. The reaction rate observed in the presence of 1 mM metal in neutral solution surpasses the rate of background hydrolysis by a factor of 10(12)-10(13) for BNPP and 10(10) for HPNPP. The increased catalytic activity is attributed principally to the preferable solvation of lanthanide ions by DMSO, which creates an anhydrous microenvironment favorable for reaction in the coordination sphere of the catalyst. The catalytic activity of lanthanides in paraoxon hydrolysis is much lower with the estimated efficiency of catalysis about 10(5) for 1 mM La(III).     

Probing the origin of the compromised catalysis of E. coli alkaline phosphatase in its promiscuous sulfatase reaction

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.     

Studies on orally active cephalosporin esters. II. Chemical stability of pivaloyloxymethyl esters in phosphate buffer solution

The degradation kinetics of pivaloyloxymethyl (POM) esters of cephalosporins in phosphate buffer solution (pH 6-8) were investigated. The degradation of the starting delta 3 cephalosporin ester proceeded mainly via isomerization to the delta 2 ester and subsequent hydrolysis to the delta 2 acid. Hydrolysis to the delta 3 acid (the parent acid) was very slow. Analysis of the rate constants indicated that the isomerization rate k12 was approximately equal to the apparent degradation rate of the delta 3 ester kdeg, and slower than the hydrolysis rate of the delta 2 ester k24. The isomerization process to the delta 2 ester was found to be the rate-determining step in the degradation of cephalosporin esters. The substituent at the C-3 position of the cephalosporins affected the degradation kinetics. The degradation was accelerated by increase of pH, buffer concentration and added protein.     

Solvent effects on the reaction coordinate of the hydrolysis of phosphates and sulfates: application of Hammond and anti-Hammond postulates to understand hydrolysis in solution.

The mechanism of the alkaline hydrolysis of phosphate and sulfate esters is of great interest. Ab initio quantum mechanical calculations and dielectric continuum methods are used to investigate the effect of the solvent on the associative/dissociative and the in-line/sideways character of the hydrolysis reaction of ethylene sulfate (ES) and ethylene phosphate (EP(-)), and their acyclic counterparts, dimethyl sulfate (DMS) and dimethyl phosphate (DMP(-)). The gas-phase reaction coordinates are determined by Hartree-Fock and density functional theory. For ES, the reaction coordinate in solution is determined; for the other three reactions only the transition state in solution is obtained. The alterations in the reaction induced by solvent are interpreted by use of the Hammond and anti-Hammond postulates.     

A one-pot coupling/hydrolysis/condensation process to pyrrolo[1,2-a]quinoxaline

CuI/L-proline-catalyzed coupling of 2-halotrifluoroacetanilides with pyrrole-2-carboxylate esters in DMSO at 80-90 degrees C followed by in situ hydrolysis at 60 degrees C afforded pyrrolo[1,2-a]quinoxalines. Indole-2-carboxylate esters underwent the same process smoothly to provide the corresponding tetracyclic products.     

A combined parallel synthesis and screening of macrocyclic lanthanide complexes for the cleavage of phospho di- and triesters and double-stranded DNA

A parallel synthesis of macrocyclic lanthanide-ligand complexes 4Ln has been developed in conjunction with a parallel screening of these ligands for catalysis of phosphate ester hydrolysis. Complexes 4Ln were screened on a 96-well plate reader for their ability to catalyze the hydrolysis of a variety of phosphate esters efficiently. The hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) 5 and p-nitrophenylethyl phosphate 6 was accelerated by up to 150-fold in the presence of the complex 4cGd. The cleavage of a double-stranded DNA plasmid with this same complex obeyed saturation kinetics following a Michaelian model (K(m) = 7.4 microM, kcat = 4.5 x 10(-3) min-1). Our findings demonstrate how a combination of parallel synthesis and screening can expedite compound access, accelerate catalyst identification, and thereby dramatically increase the speed of finding good ligand-metal combinations.     

The effect of ultrasound on the alkaline hydrolysis of nitrophenyl esters

Ultrasound was found to increase the rate of hydrolysis of a series of esters by up to 15%. No effect of molecular structure upon this enhancement was observed.